Cyclic AMP-dependent protein kinase (PKA) is anchored at specific subcellular sites through the interaction of the regulatory subunit (R) with A-Kinase Anchoring Proteins (AKAPs) via an amphipathic helix binding motif. Synthetic peptides containing this amphipathic helix domain competitively disrupt the binding of PKA to AKAPs and cause loss of PKA modulation of both glutamate receptor channels and voltage-gated calcium channels. In this report, we used S-HT31, a cell permeant anchoring inhibitor peptide (AIP), to study the role of PKA anchoring in sperm. Sperm RII and its anchoring proteins were detected by Western blot and RII overlay analysis. Sperm motility was quantitated by a computerized sperm motility analysis system (CASMA). Sperm calcium was measured by radioactive calcium uptake and Fura 2 fluorescence. Addition of S-HT31 inhibits motility of bovine caudal epididymal, ejaculated rhesus monkey and human sperm in a time- and concentration-dependent manner. A control peptide, S-HT31P, identical to S-HT31 in all respects except with a proline residue substitution to prevent amphipathic helix formation, was ineffective. Cell permeable inhibitors of the catalytic subunit of PKA also had no effect on motility. Thus interaction of the regulatory subunit of PKA with its AKAP may be critical for motility. RII` is found on the outer sperm mitochondrial membrane. Bovine sperm also contain one AKAP (110 kDa) associated with sperm midpiece, suggesting that mitochondria are involved in regulation of motility.